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Abstract:

The invention provides a method for increasing the bioactivity (e.g. the
biosafety and efficacy) of a therapeutic IgE antibody of the invention in
the treatment of a patient. Methods of the invention include: i)
administering to the patient a therapeutic IgE antibody in combination
with at least one bioactivity-enhancing agent, ii) strategic treatment
regimens and protocols for the dosing and administration of a therapeutic
IgE antibody of the invention, and iii) the use of a therapeutic IgE
antibody having a variable region comprising at least one antigen binding
region specific for binding an epitope of an antigen wherein the epitope
is not highly repetitive or is non-repetitive.

Claims:

1. A method for increasing the bioactivity of a therapeutic IgE antibody
in a patient comprising administering to the patient a therapeutic IgE
antibody in combination with at least one bioactivity-enhancing agent
selected from the group consisting of: immunostimulatory compounds;
chemotherapeutic agents; immunosuppressive agents; and any combination
thereof, in an amount effective to increase the bioactivity of the
therapeutic IgE antibody as compared to the administration of the
therapeutic IgE antibody alone.

4. The method of claim 2 wherein the IgE antibody is a chimeric antibody,
a humanized antibody or a fully human antibody.

5. The method of claim 1 wherein the immunostimulatory compound is a TLR3
agonist or a TLR4 agonist.

6. The method of claim 1 wherein the immunosuppressive agent is a
corticosteroid.

7. The method of claim 1 wherein the chemotherapeutic agent is
gemcitabine, cyclophosphamide, topotecan, or doxorubicin.

8. The method of claim 1 wherein the IgE antibody is a monoclonal
antibody comprising human Fc epsilon (ε) constant regions and a
variable region comprising at least one antigen binding region specific
for binding an epitope of an antigen wherein the epitope is not highly
repetitive or is non-repetitive.

9. The method of claim 1 wherein the bioactivity-enhancing agent is
administered to the patient at least 30 minutes prior to administration
of the therapeutic IgE antibody.

10. The method of claim 1 wherein the bioactivity-enhancing agent is
administered to the patient about 30 minutes to about 8 hours prior to
administration of the therapeutic IgE antibody.

11. The method of claim 1 wherein increased bioactivity is the reduction
or elimination of systemic hypersensitivity in the patient to the
therapeutic IgE antibody.

12. The method of claim 1 wherein increased bioactivity is the
enhancement of the patient's Th1-type immune response to the antigen.

13. The method of claim 1 wherein increased bioactivity is the
enhancement of the cytotoxic T-lymphocyte response to the antigen.

14. The method of claim 1 wherein increased bioactivity is the inhibition
of non-IgE mediated factors in the participation of a systemic allergic
response to the therapeutic IgE antibody.

15. The method of claim 1 wherein increased bioactivity is the reduction
of the dosage of the therapeutic IgE antibody or reduction in the
frequency of treatment with the antibody.

16. A method for increasing the bioactivity of a therapeutic IgE antibody
toward a tumor in a patient comprising administering to the patient a
therapeutic IgE antibody specific for an antigen associated with the
tumor in combination with at least one bioactivity-enhancing agent
selected from the group consisting of: immunostimulatory compounds,
chemotherapeutic agents, immunosuppressive agents, and any combination
thereof, in an amount effective to increase the bioactivity of the
therapeutic IgE antibody as compared to the administration of the
therapeutic IgE antibody alone.

18. The method of claim 16 wherein increased bioactivity is selected
from: inducing local hypersensitivity reactions at the site of the tumor
or in the tumor microenvironment; reduction in the dosage of the
antibody; reduction in allergic responses to the therapeutic IgE
antibody; reduction or inhibition of non-IgE mediated factors that
participate in allergic responses to the therapeutic IgE antibody;
reduction of the dosage of the therapeutic IgE antibody; and reduction in
the frequency of treatment with the IgE antibody.

19. A method of increasing the biosafety of a therapeutic IgE monoclonal
antibody in a patient comprising administering a therapeutic IgE
monoclonal antibody wherein the IgE antibody comprises a variable region
comprising at least one antigen binding region specific for binding an
epitope of an antigen wherein the epitope is not highly repetitive or is
non-repetitive optionally in combination with a bioactivity enhancing
agent in an amount effective to increase the biosafety of the IgE
monoclonal antibody as compared to the biosafety of the IgE antibody when
administered alone.

20. The method of claim 19 wherein increased biosafety is selected from:
(i) reduction of the dosage of the antibody, (ii) reduction in the
frequency of treatment with the antibody, (iii) reduction or elimination
of the occurrence of systemic hypersensitivity in a patient, (iv) induce
shifting the dominant immune response in a patient from a Th2 to Th1
immune response; and (v) reduction or inhibition of non-IgE mediated
factors that participate in allergic responses to the therapeutic IgE
antibody.

21. The method of claim 19 wherein the IgE antibody is a monoclonal
antibody selected from: a chimeric antibody; a humanized antibody; and a
fully human antibody.

22. The method of claim 19 wherein the bioactivity-enhancing agent is
administered to the patient at least 30 minutes prior to administration
of the therapeutic IgE antibody.

23. The method of claim 19 wherein the bioactivity-enhancing agent is
administered to the patient about 30 minutes to about 8 hours prior to
administration of the therapeutic IgE antibody.

24. A method of increasing the bioactivity of a therapeutic IgE antibody
comprising the step of administering an IgE antibody to a patient in an
amount effective to induce direct IgE antibody mediated toxicity against
the antigen and diseased cells and induce antigen processing and cross
presentation to induce Tc1 T-cell mediated cellular immune response to
the antigen in a patient capable of mounting such responses.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of International Application No.
PCT/US2009/040090, which designated the United States and was filed on
Apr. 9, 2009, published in English, which claims the benefit of U.S.
Provisional Application Nos. 61/043,690, filed Apr. 9, 2008, 61/044,581,
filed Apr. 14, 2008 and 61/160,157, filed on Mar. 13, 2009. The entire
teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The newly arising field of AllergoOncology is based upon
observations and studies showing that those individuals with raised
levels of IgE (e.g. individuals who suffer from allergies) are much less
likely to suffer from certain types of cancer. Researchers in this field
are exploring the therapeutic potential of the IgE antibody class in the
prevention and treatment of certain cancers.

[0004] While B cells can recognize antigen in its native conformation, T
cells generally recognize antigen that has been "processed" by antigen
presenting cells (APCs) and then presented on the surface of the cell by
major histocompatibility complex (MHC) molecules (Peakman, M. and
Vergani, D., New York: Churchhill Livingston; (1997)). MHC molecules are
receptors for peptide antigens. There are two classes of MHC molecules,
termed MHC class I and MHC class II. Although united in their function of
peptide antigen presentation and contact points for T cells, the
differences in the structure and intracellular trafficking of the two
types are critical because among other things, they elicit very different
immune responses. A major obstacle in the creation of effective tumor
immunity is that typically, there is poor presentation of tumor antigen
on MHC class I and class II molecules together (cross-presentation).
Dendritic cells are bone marrow-derived leukocytes that are more potent
initiators of T cell-dependent immune responses than any other antigen
presenting cells that have been tested (Peakman, M. and Vergani, D., New
York: Churchhill Livingston (1997)). Unlike other APCs, dendritic cells
can acquire antigens from their environment and process them for
cross-presentation, allowing activation of both CD8.sup.+ and CD4.sup.+ T
cells. However, this process requires high antigen concentrations.
Simultaneous presentation on MHC II provides for T helper cell
activation. Depending on the stimuli, either production of cytokines
IL-12 and IFN-γ by T helper (Th) cell 1 type and cytotoxic
T-lymphocyte (CTL) induction occurs (collectively referred to herein as
the "Th1/Tc1 immune response); or IL-4, IL-5 and IL-10 is produced by Th2
cells for B cell help (referred to herein as "Th2 immune response"). An
important factor in immune induction is the activation or maturation of
the APC, which induces the expression of co-stimulatory molecules that
are necessary to engage the T cell.

[0006] IgE binds to two types of Fc receptors, called FcεRI (or
high-affinity FcεR) (Ka=1011 M-1) and
FcεRII (or low-affinity FcεR, CD23) (Ka<108
M-1). Therefore, unlike antibodies of the IgG class, IgE binds to
its FcεRI with extremely high affinity which in the case of
FcεRI is about 3 orders of magnitude higher than that of IgG for
the FcRs (FcγRI-III) and in the case of FcεRII is similar
to the affinity of IgG for its high affinity FcγRI (Gould, H J, et
al., Annu. Rev. Immunol., 21: 579-628. Epub@2001 December@19:579-628
(2003); Gounni, A S, et al., Nature, 367: 183-186 (1994); Kinet, J P,
Annu. Rev. Immunol., 17: 931-972 (1999) and Ravetch J V, and Kinet J P,
Annu. Rev. Immunol., 9: 457-492 (1991)). Because the IgE concentration in
normal serum is usually very low (less than 1 μg/mL), the FcεR
are typically available for occupancy if IgE is induced by allergies and
parasitic infestation or if administered. The FcεRI is composed
of four polypeptide chains, one α, one β, and two γ
chains. The α chain contains the IgE binding site and is a member
of the immunoglobulin supergene family. The FcεRII consists of
one polypeptide chain which shows homology to animal lectin receptors.
FcεRI is expressed on mast cells and basophils as well as
Langerhans cells and dendritic cells where it is involved in antigen
presentation, on eosinophils where it plays a role in defense against
parasitic infection, and also on monocytes (see Kinet, J P, Annu. Rev.
Immunol., 17: 931-72:931-972 (1999) for a review). Crosslinking of the
FcεRI receptors via bridging of bound IgE induces immediate
release of mediators of inflammation such as histamine, various cationic
proteases, leukotrienes, prostaglandin E2, or β-glucuronidase, and
delayed secretion of IL-4, 5, and 6. FcεRII is a member of the Ig
superfamily, more widely expressed on resting and mature B cells,
monocytes, follicular dendritic cells, macrophages, eosinophils,
platelets, Langerhans cells, and a subset of T cells (10-15% of tonsillar
T cells). IL-4 up-regulates FcεRII expression on B cells and
macrophages. FcεRII on macrophages, eosinophils, and platelets
mediates ADCC to schistosomules, enhance phagocytosis, and induce the
release of granule enzymes (Gounni, A S, et al., Nature, 367: 183-186
(1994); Kinet, J P, Annu. Rev. Immunol., 17: 931-972 (1999) and
Spiegelberg, H L, J. Invest. Dermatol., 94: 49S-52S (1990)).
FcεRII is involved in both IgE regulation and allergen
presentation by B-cells, but understanding the functional roles of CD23
is further complicated by the fact that it exists both as a cell surface
molecule and in a soluble form generated by cleavage from the cell
surface; furthermore, it exists in both monomeric and oligomeric states
(see Gould, H J, and Sutton, B J, Nat. Rev. Immunol., 8:205-217 (2008)
for a review). CD23 responds to high levels of IgE by downregulating IgE
secretion. In human monocytes, CD23 triggering results in release of
pro-inflammatory cytokines including tumor necrosis factor (TNF)-α,
IL-1, IL-6, and granulocyte/macrophage-colony stimulating factor
(GM-CSF). IL-4 appears to play a central role in immediate-type
hypersensitivity. It induces human B cells to secrete IgE and IgG4 and
activated T helper cells. IL-4 also stimulates mast cell growth and
up-regulates FcεRII expression.

[0007] Most of the antibodies used in the treatment of cancer, including
FDA approved antibodies such as trastuzumab (HERCEPTIN®) and
rituximab (RITUXAN®)), are of the IgG class (Carter, P., IBC's Tenth
International Conference. 6-9 Dec. 1999, La Jolla, Calif., USA. IDrugs.
3:259-261 (2000); Carter, P., Nat. Rev. Cancer, 1: 118-129 (2001) and
Carter, P J, Nat. Rev. Immunol., 6: 343-357 (2006)). However, four
monoclonal IgE antibodies specific for tumor antigens have been reported.
The application of IgE for the therapy of cancer was pioneered by Nagy et
al. (Nagy, E., et al., Cancer Immunol. Immunother., 34: 63-69 (1991)),
who developed a murine IgE monoclonal antibody specific for the major
envelope glycoprotein (gp36) of mouse mammary tumor virus (MMTV) and
demonstrated significant anti-tumor activity in C3H/HeJ mice bearing a
syngeneic MMTV-secreting mammary adenocarcinoma (H2712) (Nagy, E., et
al., Cancer Immunol. Immunother., 34: 63-69 (1991)). Kershaw et al.
(Kershaw, M H, et al., Oncol. Res., 10: 133-142 (1998)) developed a
murine monoclonal IgE named 30.6, specific for an antigenic determinant
expressed on the surface of colorectal adenocarcinoma cells. Mouse IgE
30.6 inhibited the growth of established human colorectal carcinoma COLO
205 cells growing subcutaneously in severe combined immune deficient
(SCID) mice, although this effect was transient. By contrast, a mouse IgG
30.6 and a mouse/human chimeric IgE 30.6 did not show anti-tumor effects.
The mouse IgE specific effect was attributed to the interaction of the
antibody with FcεR bearing effector cells since the activity was
specifically abrogated by prior administration of a nonspecific mouse IgE
(Kershaw, M H, et al., Oncol. Res., 10: 133-142 (1998)). The lack of
effect exhibited by the mouse/human chimeric IgE 30.6 is explained by the
fact that mouse FcεRI binds mouse IgE, but not human IgE. Gould
et al. (Gould, H J, et al., Eur. J. Immunol., 29: 3527-3537 (1999))
developed a mouse/human chimeric IgE (MOv18-IgE) and IgG MOv18 (IgG1)
specific for the ovarian cancer tumor associated antigen folate binding
protein (FBP). The protective activities of MOv18-IgE and MOv18-IgG1 were
compared in a SCID mouse xenograft model of human ovarian carcinoma
(IGROV1). Mice were reconstituted with human peripheral blood mononuclear
cells (PBMC) to provide the model with effector cells capable of binding
human IgE constant regions. The beneficial effects of MOv18-IgE were
greater and of longer duration than those of MOv18-IgG1 demonstrating the
superior anti-tumor effects of IgE antibodies (Gould, H J, et al., Eur.
J. Immunol., 29: 3527-3537 (1999)). In addition, the group of Gould et
al. recently demonstrated for the first time monocyte-mediated
IgE-dependent tumor cell killing by two distinct pathways, ADCC and
phagocytosis (ADCP), mediated through FcεRI and FcεRII
(Karagiannis, S N, et al., Cancer Immunol. Immunother., 57: 247-263
(2008) and Karagiannis, S N, et al., J. Immunol., 179: 2832-2843 (2007)).
This group has also used this assay system to make a preliminary
assessment of bioactivity of an anti-Her2 IgE construct (Karragiannis,
P., Cancer Immunol and Immunother (2008) epub ahead of print). Since
human PBMC are short-lived in SCID mice the inventors have postulated
that the anti-tumor effect will be enhanced in humans where the supply of
effector cells would be permanent. None of the studies could address the
capacity of the mouse/human chimeric IgE to elicit an adaptive immune
response due to the fact that murine APCs such as dendritic cells do not
express the FcεRI (Kinet, J P, Annu. Rev. Immunol., 17:
931-72:931-972 (1999)).

[0009] Furthermore, mice infested with nematodes are resistant to
syngeneic mammary adenocarcinoma and show lower incidence of spontaneous
mammary tumors (Ogilvie, B M, et al., Lancet., 1: 678-680 (1971) and
Weatherly, N F, J. Parasitol., 56: 748-752 (1970)). Eosinophilia, either
in peripheral blood or tumor-associated tissue, is frequently associated
with some tumor types and also found after immunotherapy with IL-2, IL-4,
GM-CSF, and antibody to CTLA-4 (Lotfi, R, et al., J. Immunother., 30:
16-28 (2007). Within several tumor types including gastrointestinal
tumors, this observation is associated with a significantly better
prognosis, whereas their presence in rejecting allografts is largely seen
as a harbinger of poor outcome (Lotfi, R. and Lotze, M T, J. Leukoc.
Biol., 83: 456-460 (2008)). Matta et al. (Clin Cancer Res 13: 5348-5354
2007) have reported that multiple myeloma patients with relatively higher
IgE levels had a better survival than patients with lower levels of IgE.
Importantly, this is clearly reflected on the levels of IgE and not the
other classes of immunoglobulins. These studies are consistent with a
natural role of IgE in the immunosurveillance of cancer including
multiple myeloma. Fu, et al. (Clin Exp Immunol 153: 401-409 (2008))
demonstrated that antibodies of the IgE class isolated from pancreatic
cancer patients mediate antibody-dependent cell-mediated cytotoxicity
against cancer cells.

[0010] Finally, treatment with omalizumab (XOLAIR®), which decreases
free IgE in serum and down-regulates IgE receptors in effector cells to
dampen IgE-mediated inflammatory response, appears to lead to a higher
chance of developing cancer. Approximately 1 in 200 treated asthmatic
patients developed breast, prostate, melanoma, non-melanoma skin, or
parotid gland malignancies during the median observation period of 1 year
while in the control group the incidence was 1 in 500 (Dodig, S., et al.,
Acta Pharm., 55: 123-138 (2005)). These studies suggest a natural role of
IgE in the immunosurveillance of cancer.

[0011] The art has established methods to treat patients who have
developed hypersensitivity reactions to chemotherapeutic agents as well
as monoclonal antibodies used in the treatment of autoimmune disease and
malignancy in which a rush desensitization to the therapeutic agent is
performed (Castells et al., J. Allergy Clin. Immunol. (2008) 122:574).
Castells describes a protocol that reduces immunogenicity to an IgG class
therapeutic antibody by administering increasing amounts of
subtherapeutic dosages to achieve desensitization to the IgG therapeutic
over a 4-8 hour period. It is noteworthy that the typical starting
concentration for a desensitization protocol with Rituxan is 0.034 mg/mL
reflecting the high antibody doses required to achieve clinical effects
with IgG1 class cancer targeting antibodies. The art does not address the
use of IgE monoclonal antibodies as therapeutic agents or methods for
mitigating hypersensitivity reactions when IgE monoclonals are used.

SUMMARY OF THE INVENTION

[0012] The invention provides methods for increasing the bioactivity (e.g.
biosafety and/or efficacy) of a therapeutic IgE antibody in the treatment
of a patient comprising administering to the patient a therapeutic IgE
antibody in combination with at least one bioactivity-enhancing agent
preferably selected from the group consisting of immunostimulatory
compounds, chemotherapeutic agents, immunosuppressive agents, and any
combination thereof, in an amount effective to increase the bioactivity
of the therapeutic IgE antibody as compared to the bioactivity of the
therapeutic IgE antibody when administered alone.

[0013] The invention further provides methods for increasing the
bioactivity and particularly the biosafety of a therapeutic IgE antibody
of the invention comprising administering to the patient a therapeutic
IgE antibody of the invention comprising Fc epsilon (ε) constant
regions and a variable region comprising at least one antigen binding
region specific for binding an epitope of an antigen wherein the epitope
is not highly repetitive or is non-repetitive.

[0014] The invention further provides methods for increasing the
bioactivity of a therapeutic IgE antibody through the use of strategic
treatment regimens and protocols for the dosing and administration of a
therapeutic IgE antibody of the invention optionally in combination with
at least one bioactivity enhancing agent. For example, the inventors are
the first to appreciate that not only can therapeutic IgE antibodies be
dosed in a much lower range than IgG antibodies, but also that the dosage
range of a therapeutic IgE antibody which is effective to induce a potent
direct IgE antibody mediated toxicity against the antigen and diseased
cells is also effective for antigen processing, cross presentation and
specific T cell stimulation of adaptive cellular immunity. The IgG
therapeutic antibodies of the prior art lack this advantage. Typically
the dosage of an IgG therapeutic antibody required to elicit the desired
effector cell response against the target antigen is orders of magnitude
higher than the dosage required for effective antigen cross presentation
and T cell stimulation mediated by the IgG therapeutic antibody. Thus,
what is considered the appropriate therapeutic dosage of a therapeutic
IgG antibody is not the optimal dosage for also eliciting an antigen
specific T cell response by the therapeutic IgG antibody and rather is
inhibitory for antigen specific cross presentation and T cell
stimulation. Monoclonal IgE can uniquely utilize effector cell mediated
and specific T cell mediated immune pathways at a common dose.

[0015] The invention further provides methods for increasing the
bioactivity, particularly the biosafety, of a therapeutic IgE antibody of
the invention by enhancing the Th1-type immune response and CTL immune
response (collectively referred to herein as the "Th1/Tc1" immune
response) to an immune complex comprising an antigen and a therapeutic
IgE antibody, comprising administering to the patient capable of mounting
such Th1/Tc1 immune response, a therapeutic monoclonal IgE antibody
optionally in combination with at least one bioactivity-enhancing agent.

DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1: schematic of antigen presentation assay (APA). Dendritic
cells (DC) are cultured from human PBMCs in the presence of IL-4 and
GM-CSF. On day 7, the primary culture is loaded with tumor associated
antigen (TAA) and antibody (Ab) and matured with a standard maturation
cocktail (e.g. TNF-α/IFN-α). Next, T cells are added and
cultured for 7 days. Subsequently, the T cell culture is stimulated with
two additional rounds of TAA/Ab using fresh DC cultures. Following the
third round of TAA/Ab stimulation, the T cells are analyzed for
tumor-specific responses.

[0017] FIG. 2: antigen presentation is enhanced by anti-Her2 IgE and IgG1
Abs. T cells from APA assay were analyzed for IFN-γ production by
intracellular cytokine staining and flow cytometry. T cells from the APA
were stained with anti-CD3-FITC and anti-CD8-PECy5 and then permeabilized
and stained with anti-IFNγ-PE. Plots are gated on CD3 (FITC)
positive cells and show IFN-γ vs CD8 staining The top panel is
plots of T cells stimulated with PMA and ionomycin (positive control) or
T cells left in media alone (negative control). The bottom panel includes
plots of T cells that were stimulated in the APA with ECD-Her2 alone (no
Ab) or with anti-Her-2 IgE or anti-Her2 IgG1.

[0018]FIG. 3: antigen presentation is enhanced by anti-Her2 IgE and IgG1
Abs. Bar graph of the results of the antigen presentation assay with
anti-HER-2 IgE and anti-HER-2 IgG1. Values are the percentage of CD8+ and
CD8- (CD4+) cells within the live CD3+ gate that are positive for
intracellular IFN-γ staining.

[0019] FIG. 4: Antigen presentation is enhanced by anti-Her2 IgE and IgG1
Abs. Bar graph of the results of the antigen presentation assay with
anti-HER-2 IgE and anti-HER-2 IgG1. Values are the percentage of CD8+ and
CD8- (CD4+) cells within either the live CD3+ CD8+ gate or the live CD3+
CD8- (CD4+) gate that are positive for intracellular IFN-γ
staining.

[0021] The expectation that IgE will mediate potentially life threatening
allergic reactions if infused into patients is the prejudice that
currently exists in the state of the art. The present invention provides
methods for improving the safety and bioactivity of IgE antibodies to
direct hypersensitivity reactions for the purpose of controlling a
disease state and altering antigen processing of disease specific
antigens (e.g. tumor antigens and other antigen sources) while avoiding
systemic hypersensitivity reactions.

[0022] While not be limited to any particular scientific theory, it is
believed that monoclonal IgE antibodies of the invention are capable of
directing hypersensitivity reactions by binding local disease related
antigens either on the surface of the diseased tissue or in the
microenvironment and bringing Fc epsilon R1 and R2 bearing effector cells
including mast cells, basophils, macrophages and eosinphils to the site
of a disease process (e.g. a micrometastatic focus of a malignant tumor,
or the site of a chronic infection (e.g. tuberculin nodule, virally
infected hepatocyte) inducing a local allergic inflammatory reaction with
local cellular activation in the absence of systemic anaphylaxis.

[0023] It is also believed that IgE antibodies are capable of altering
antigen processing to cancer and other diseases by binding disease
associated antigen in circulation or at the site of a disease process
(e.g. a tumor site) and being taken up through FcεRII mediated
antigen processing; resulting in cross presentation of antigen peptide
fragments in the context of MHC class I and MHC class II resulting in a
robust cell mediated immune response including both CD4+ and CD8+ T cell
activation, with a Tc1 dominance.

[0024] While the inventors have appreciated that both of these processes
can be mediated by monoclonal IgE, despite the teaching of the art that
would suggest a Th2 immune response would dominate; this effect can best
be accomplished by careful selection of dose and administration of
monoclonal IgE antibody and optionally with the selection and timing of
co-administered agents to prevent systemic reactions to further boost the
desired specific immune effector responses in the face of endogenous
counter-regulatory pathways extant to limit autoimmunity In one
embodiment, antibodies comprising Fc epsilon (ε) constant regions
and a variable region comprising at least one antigen binding region
specific for an antigen (e.g. a cancer antigen), when optionally
administered with an bioactivity-enhancing agent, will not only induce a
Th2-type immune response to the antigen in a patient but will also
enhance the Th1-type immune response to the antigen in a patient
including enhancement of the CTL response and also the humoral immune
response mediated by B cells. In one embodiment, the methods of the
invention may induce shifting the dominant immune response in a patient
from a Th2 to Th1 immune response (also referred to herein as "enhancing
the Th1/Tc1 immune response"). The effect of such a shift would be to
reduce the chance of immediate systemic hypersensitivity in a patient
through in enrichment of IFN gamma antigen specific T cells and
enhancement of cellular immune pathways. Additionally or alternatively,
the methods of the invention may reduce or eliminate systemic
hypersensitivity by reducing vigor of crosslinking of Fcε
receptors via the choice of non repetitive epitope targets in the
production of IgE and/or through precise co-medication to prevent the
elicitation of allergic inflammatory histamine releasing factors that
promote systemic not limited local immediate hypersensitivity reactions
(mast cell degranulation).

[0025] A "therapeutic IgE antibody" is an antibody comprising Fc epsilon
(ε) constant regions and a variable region comprising at least
one antigen binding region specific for an antigen (e.g. a cancer
antigen), that can bind to an antigen on a target cell or in circulation
to cause a therapeutic effect in a patient. In one embodiment, the
antigen is not an allergen or other antigen that is the normal
physiological target of unmodified IgE present in the subject. In a
preferred embodiment, the therapeutic IgE antibody is a monoclonal
antibody.

[0026] The terms "monoclonal antibody" or "monoclonal antibodies" as used
herein refer to a preparation of antibodies of single molecular
composition. A monoclonal antibody composition displays a single binding
specificity and affinity for a particular epitope. The monoclonal
antibodies of the present invention are preferably chimeric, humanized,
or fully human in order to bind human Fc epsilon receptors when the
subject host is a human. Humanized and fully human antibodies are also
useful in reducing immunogenicity toward the murine components of, for
example, a chimeric antibody, when the host subject is human. Methods for
producing monoclonal antibodies are well known in the art.

[0027] In one preferred embodiment, the therapeutic IgE antibody is a
chimeric monoclonal antibody. The term "chimeric monoclonal antibody"
refers to monoclonal antibodies displaying a single binding specificity
which have one or more regions derived from one antibody and one or more
regions derived from another antibody. In a preferred embodiment of the
invention, the constant regions are derived from human Fc epsilon
(ε) (heavy chain) and human kappa or lambda (light chain)
constant regions. The variable regions of a chimeric antibody may be of
human or non-human origin but are typically of non-human origin. In one
embodiment, the variable region is of non-human origin such as from
rodents, for example, mouse (murine), rabbit, rat or hamster. In one
embodiment, the variable region is of murine origin. Previously published
methodology used to generate mouse/human chimeric or humanized antibodies
that has yielded the successful production of various human chimeric
antibodies or antibody fusion proteins (Helguera G, Penichet M L.,
Methods Mol. Med. 109:347-74 (2005)). Other methods for producing
chimeric antibodies are well known in the art.

[0029] Fully human or human-like antibodies may be produced through
vaccination of genetically engineered animals such as mouse lines
produced at Abgenix (CA) and MedaRex (NJ) which contain the human
immunoglobulin genetic repertoire and produce fully human antibodies in
response to vaccination. Further, the use of phage display libraries
incorporating the coding regions of human variable regions which can be
identified and selected in an antigen screening assay to produce a human
immunoglobulin variable region binding to a target antigen.

[0030] In one embodiment, the therapeutic IgE antibody is not a chimeric
antibody comprising a human Fcε constant region and mouse
variable region having an antigen binding region that is specific for an
epitope of HER2 (which binds the epitope of HER2 defined by the antibody
contained in Herceptin) or an antigen binding region that is specific for
an epitope of CD20 (which binds the epitope of CD20 that is defined by
the antibody contained in Rituximab or an antigen which binds an epitope
specific for an antibody disclosed in U.S. Pat. No. 5,977,322.

[0031] The term "antigen binding region" refers to that portion of an
antibody of the invention which contains the amino acid residues that
interact with an antigen and confer on the antibody its specificity and
affinity for the antigen. The antibody region includes the "framework"
amino acid residues necessary to maintain the proper confirmation of the
antigen binding residues.

[0032] An "antigen" is a molecule or portion of a molecule capable of
being bound by an antibody which is additionally capable of inducing an
animal to produce antibody capable of binding to an epitope of that
antigen. In one embodiment, the antigen is capable of being bound by an
IgE antibody of the invention to form an immune complex that is capable
of inducing a specific IgE-mediated immune response to the antigen in a
patient capable of mounting such immune response. As used herein, a
"patient capable of mounting (the referenced) immune response" is a
subject such as a human patient or other animal subject with functional
T-cells, mast cells, basophils, eosinophils, monocytes, macrophages and
dendritic cells with receptor affinity for the administered IgE antibody
of the invention as distinguished from non-human animal models, for
example, whose immune systems do not contain Fc epsilon receptors capable
of binding human IgE permitting generation of functional T-cells, mast
cells, eosinophils and dendritic cells in response to the administered
antibody.

[0033] Preferred antigens include any soluble antigen that is detectable
in body fluid (e.g. blood serum ascites, saliva or the like). In one
preferred embodiment, the antigen is a tumor associated antigen (TAA). In
one embodiment, the antigen, on its own, may not be capable of
stimulating an immune response or elicits only a weak immune response for
any number of reasons, for example, the antigen is a "self" antigen, not
normally recognized by the immune system as requiring response or the
immune system has otherwise become tolerant to the antigen and does not
mount an immune response.

[0034] An antigen can have one or more epitopes that are the same or
different. In one embodiment, the antibodies of the invention are
specific for a single, non-repetitive epitope of the antigen. Thus, the
immune complex formed by an antibody of the invention and its antigen is
referred to as "monovalent" in that only one antibody of the invention
may be bound to a single molecule of antigen at any one time.

[0035] The term "epitope" is meant to refer to that portion of any
molecule capable of being recognized by and bound by an antibody at one
or more of the antibody's binding regions. Epitopes generally comprise
chemically active surface groupings of molecules such as amino acids or
sugar side chains and have specific three dimensional structure
characteristics as well as specific charge characteristics. The term
"non-repetitive epitope" means that only one such epitope is present in
the antigen. An epitope that is not "highly repetitive" means an epitope
whose frequency and configuration upon the antigen are such that when an
immune complex is formed between the therapeutic IgE antibody and the
antigen, such immune complex does not cause crosslinking of the
Fcε receptors on dendritic cells or other relevant APCs.

[0036] An "immune complex" (IC) is a complex formed by an antibody and its
target antigen. An immune complex may be "polyvalent" meaning that more
than one antibody is associated with an antigen, or "multimeric" meaning
that multiple antigens and antibodies are complexed together, or
"monovalent" meaning that each antigen molecule is bound to only one
antibody molecule.

[0037] The term "cancer antigen" as used herein can be any type of cancer
antigen known in the art. The cancer antigen may be an epithelial cancer
antigen, (e.g., breast, gastrointestinal, lung), a prostate specific
cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a
bladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, a
colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen,
a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic
cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a
head and neck cancer antigen, or a colorectal cancer antigen. A cancer
antigen can also be a lymphoma antigen (e.g., non-Hodgkin's lymphoma or
Hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemia
antigen, a myeloma (i.e., multiple myeloma or plasma cell myeloma)
antigen, an acute lymphoblastic leukemia antigen, a chronic myeloid
leukemia antigen, or an acute myelogenous leukemia antigen. Other cancer
antigens include but are not limited to mucin-1 protein or peptide
(MUC-1) that is found on all human adenocarcinomas: pancreas, colon,
breast, ovarian, lung, prostate, head and neck, including multiple
myelomas and some B cell lymphomas; mutated B-Raf antigen, which is
associated with melanoma and colon cancer; human epidermal growth factor
receptor-2 (HER-2/neu) antigen; epidermal growth factor receptor (EGFR)
antigen associated lung cancer, head and neck cancer, colon cancer,
colorectal cancer, breast cancer, prostate cancer, gastric cancer,
ovarian cancer, brain cancer and bladder cancer; prostate-specific
antigen (PSA) and/or prostate-specific membrane antigen (PSMA) that are
prevalently expressed in androgen-independent prostate cancers; is Gp-100
Glycoprotein 100 (gp 100) associated with melanoma carcinoembryonic (CEA)
antigen; carbohydrate antigen 10.9 (CA 19.9) related to the Lewis A blood
group substance and is associated with colorectal cancers; and a melanoma
cancer antigen such as MART-1.

[0038] In one preferred embodiment, the invention provides methods of
increasing the bioactivity of a therapeutic IgE monoclonal antibody in a
patient comprising administering a therapeutic IgE monoclonal antibody of
the invention in combination with a bioactivity enhancing agent in an
amount effective to increase the bioactivity of the IgE monoclonal
antibody as compared to the bioactivity of the IgE antibody when
administered alone. The term "bioactivity-enhancing agent" refers to an
agent preferably selected from the group consisting of immunostimulatory
compounds, chemotherapeutic agents, anti-cancer antibodies and
anti-inflammatory agents, in an amount able to increase the bioactivity
of the therapeutic IgE antibody as compared to the bioactivity of the
therapeutic IgE antibody when administered alone. "Increasing the
bioactivity of a therapeutic IgE antibody" is meant to refer to any one
or more of the following outcomes favorably impacting the therapeutic
index of the IgE antibody when administered to a patient in vitro or in
vivo (in an appropriate animal system) or ex vivo suffering from a
disease (e.g. cancer) associated with the expression of an antigen
targeted by an IgE antibody of the invention, or when used in a
preclinical model of such a clinical circumstance: (i) reduction in tumor
size, (ii) extension of time to tumor progression, (iii) extension of
disease- or tumor-free survival, (iv) increase in overall survival, (v)
reduction of the dosage of the antibody, (vi) reduction of the rate of
disease progression, (vii) increased amelioration of disease symptoms,
(viii) reduction in the frequency of treatment, (ix) reduction or
elimination of the occurrence of systemic hypersensitivity in a patient,
(x) enhancement of a Th1 type immune response in a patient, (xi)
enhancement of CTL immune response in a patient, (xii) reduction in
allergic responses to the therapeutic IgE antibody, (xiii) reduction or
inhibition of non-IgE mediated factors that participate in allergic
responses to the therapeutic IgE antibody, (xiv) activating T cells, (xv)
eliciting ADCC and ADCP immune responses in a patient capable of mounting
such a response, (xvi) mobilizing the use of macrophage, monocyte,
eosinophil, basophil, and mast cells as effector cells (xvii) inducing
local hypersensitivity reactions including ADCC, ADCP, and CTL responses
at the site of the tumor or in the tumor microenvironment in a patient
capable of mounting such a response. In certain embodiments, increased
bioactivity is compared to the bioactivity (e.g., the predicted or
measured bioactivity using appropriate pre-clinical models) of the
therapeutic IgE antibody used in a treatment without a compound (i.e. a
bioactivity enhancing agent) of the invention.

[0039] In one preferred embodiment, the invention provides methods of
increasing the biosafety of a therapeutic IgE monoclonal antibody in a
patient comprising administering a therapeutic IgE monoclonal antibody of
the invention in combination with a bioactivity enhancing agent in an
amount effective to increase the biosafety of the IgE monoclonal antibody
as compared to the biosafety of the IgE antibody when administered alone.
The term "increasing the biosafety" of an therapeutic IgE antibody means
any one or more of the following outcomes: (i) reduction of the dosage of
the antibody, (ii) reduction in the frequency of treatment, (iii)
reduction or elimination of the occurrence of systemic hypersensitivity
in a patient, (iv) induce shifting the dominant immune response in a
patient from a Th2 to Th1 immune response; (v) reduction or inhibition of
non-IgE mediated factors that participate in allergic responses to the
therapeutic IgE antibody. In certain embodiments, increased biosafety is
compared to the biosafety (e.g., the predicted or measured biosafety
using appropriate preclinical models) of the therapeutic IgE antibody
used in a treatment without a compound (i.e. a bioactivity enhancing
agent) of the invention.

[0041] In one preferred embodiment the immunostimulatory compound is a
TLR3 agonist. In preferred embodiments, the TLR3 agonist for use
according to the invention is a double stranded nucleic acid selected
from the group consisting of: polyinosinic acid and polycytidylic acid,
polyadenylic acid and polyuridylic acid, polyinosinic acid analogue and
polycytidylic acid, polyinosinic acid and polycytidylic acid analogue,
polyinosinic acid analogue and polycytidylic acid analogue, polyadenylic
acid analogue and polyuridylic acid, polyadenylic acid and polyuridylic
acid analogue, and polyadenylic acid analogue and polyuridylic acid
analogue. Specific examples of double-stranded RNA as TLR3 agonists
further include Polyadenur (Ipsen) and Ampligen (Hemispherx). Polyadenur
is a polyA/U RNA molecule, i.e., contains a polyA strand and a polyU
strand. Ampligen is disclosed for instance in EP 281 380 or EP 113 162.

[0042] In one embodiment the immunostimulatory compound is a TLR4 agonist.
Exemplary TLR4 agonists include taxanes such as paclitaxel and docetaxal,
lipopolysaccharides (LPS); E. coli LPS; and P. gingivalis LPS.

[0045] In one embodiment, the invention provides a method for increasing
the bioactivity, particularly the biosafety of an IgE antibody of the
invention comprising enhancing the Th1/Tc1 immune response to an immune
complex comprising an antigen and an IgE antibody in a patient,
preferably a human patient capable of mounting such Th1/Tc1 immune
response, comprising administering to the patient a therapeutic
monoclonal IgE antibody of the invention optionally in combination with
at least one bioactivity-enhancing agent preferably selected from the
group consisting of: immunostimulatory compounds, chemotherapeutic
agents, immunosuppressive agents, and any combination thereof, in an
amount effective to enhance the patient's Th1/Tc1 response (e.g. a CD4,
CD8 CTL, IFN gamma associated cellular response) to the immune complex.
The effect of a primary CD8 CTL response would be to mediate anti-tumor
effects with reduced tendency to induce clinically worrisome immediate
hypersensitivity in a patient such as systemic anaphylaxis. As used
herein the term "enhancing" includes switching the dominant immune
response in a subject from a Th2 response to a Th1 response.

[0046] In one embodiment, the invention provides a method for increasing
the bioactivity, particularly the biosafety of an IgE antibody of the
invention comprising administering to the patient a therapeutic IgE
antibody of the invention comprising Fc epsilon (ε) constant
regions and a variable region comprising at least one antigen binding
region specific for binding an epitope of an antigen wherein the epitope
is not highly repetitive or is non-repetitive optionally in combination
with a bioactivity enhancing agent. Additionally or alternatively, the
methods of the invention may reduce or eliminate systemic
hypersensitivity by reducing or eliminating crosslinking of Fcε
receptors which is the cause of mast cell or basophil degranulation.
Systemic reactions such as systemic anaphylaxis are generally associated
with a polyclonal IgE response, and production of T cell derived mast
cell activating factors that permit a local reaction to become systemic.
Systemic reactions are associated with the vigorous crosslinking
associated with a multi-epitopic polyclonal IgE response. Immune
complexes comprising antigen an in combination with an IgE antibody of
the invention to a non-repetitive epitope of the antigen will be less
likely to crosslink FcεRs to the level of systemic symptoms when
bound to mast cells or granulocytes in circulation than compared to
antigen specific polyclonal IgE.

[0047] Therefore, the invention also provides methods of reducing systemic
hypersensitivity in a patient being treated with a therapeutic IgE
antibody comprising administering to the patient a therapeutic IgE
monoclonal antibody of the invention comprising at least one antigen
binding region specific for binding an epitope, preferably of an antigen
wherein the epitope is not highly repetitive or is non-repetitive,
optionally in combination with at least one bioactivity-enhancing agent
selected from the group consisting of: immunostimulatory compounds,
chemotherapeutic agents, immunosuppressive agents, and any combination
thereof, in an amount effective to reduce systemic hypersensitivity in a
patient as compared to the administration of the therapeutic IgE antibody
alone.

[0048] Currently the state of the art teaches that when immune complexes
consisting of antigen and polyclonal IgE antibodies or IgE antibodies to
a multi-epitopic allergen bind to antigen presenting cells in atopic
individuals as well as to mast cells or basophils, the FcεR would
be crosslinked, which leads to cellular activation, local cytokine
production favoring a Th2 biased immune response, activation of T helper
2 (Th2) cells, and secretion of interleukin (IL)-4 and IL-5. Thus the art
expects that the cytokines will subsequently induce Th2 immunity and lead
to allergic inflammation including recruitment and activation of
eosinophils and other allergic inflammatory cells. Maurer, D., et al., J.
Immunol., 161: 2731-2739 (1998) and Maurer D., et al., J. Immunol., 154:
6285-6290 (1995). The art does not address immunity in a circumstance
where the antigen is self tumor antigen and not an allergen.

[0049] However, the inventors have found that when immune complexes
consisting of antigen and monoclonal IgE antibodies to a tumor antigen
epitope are bound by dendritic cells derived from a non atopic patient in
an in vitro cell culture system, the dendritic cells express primarily
Fcε RII and produce a T cell stimulation to the antigen that has
characteristics of a Th1/Tc1 immune response, with prominent induction of
IFN-gamma producing CD4 and CD8 antigen specific lymphocytes. Of
particular importance is the generation through this mechanism of
specific and protective CD8 IFN-gamma positive cytotoxic T-lymphocytes
(CTLs) response against the antigen which will cause lysing of tumor
cells or at metastatic sites that express the tumor antigen in context of
MHC class I.

[0050] IgE mediated mast cell and local basophil activation will result in
a local reaction that is measurable quantitatively with acute measurement
of the ensuing wheal and flare reaction (allergen skin test) in humans or
rodents and other higher organisms. The immediate skin test reaction is
only marginally inhibited in the presence of an acute dose of
corticosteroid and reflects local vascular leak and increased local blood
flow in response to histamine, prostaglandin, leukotriene, and other
acute mediators released form the allergic effector cells. Additional
late allergic symptoms also occur (Macguire, Nicodemus, Clin Immun 1999)
(Norman et al, Am J Resp Crit care Med, 1996) as observed following
administration of T cell epitope enriched, but non IgE reactive peptides.
Although the factors that definitively mediate these reactions have yet
to be established in the Art, they are not IgE mediated. Without being
limited to any one theory, it is believed therefore that inhibition of
non-IgE mediated systemic factors that play a role in the allergic immune
response using a bioactivity enhancing agent in combination with an IgE
antibody in accordance with the invention would result in the dampening
of systemic hypersensitivity while preserving local IgE mediated allergic
immune response against an antigen. Thus it is possible to use monoclonal
IgE as a safe and highly bioactive therapy to induce specific immunity,
mediate targeted cell killing, and provide a new modality for the
treatment of malignancy.

[0051] The expectation and teaching of immunotherapist has been that
corticosteroid administration is immune suppressive, as is cytotoxic
chemotherapy administration; and therefore clinically effective
immunotherapy should be given remote from such interventions. Recently
the inventors (Braly, Nicodemus, et al, JIT 2009) have demonstrated that
concomitant corticosteroid and paclitaxel/carboplatin administration can
be immune enhancing in inducing immunity with intravenously administered
xenotypic IgG to a tumor antigen in ovarian cancer patients. In addition
they reported that the schedule of component administration importantly
impacts the kinetics and magnitude of the immune responses. By extension,
the safe and effective administration of a therapeutic monoclonal IgE to
a target antigen can be accomplished through premedication with a
corticosteroid 8 hours to 30 minutes prior to antibody infusion. While
local reactions at the site of pathology will occur; and late cellular
immunity induced through uptake of IgE/antigen by antigen presenting
cells systemic anaphylaxis will be avoided. The clinical effect can be
further potentiated by following the antibody administration 1 to 8 hours
later with the infusion of a specific immune adjuvant, preferably a TLR
agonist. In the treatment of malignancy, acute cytotoxic agents can be
administered concurrently, although as the pharmacology of specific
cancer treatments varies, and advances in cancer treatment must start
with standard of care interventions, the cancer specific interventions
must be customized empirically.

[0052] The methods of the invention also comprise increasing the
bioactivity of a therapeutic IgE antibody of the invention through the
use of strategic dosing and/or administration protocols. For example, the
inventors are the first to appreciate that not only can therapeutic IgE
antibodies be dosed in a much lower range than IgG antibodies currently
in typical clinical use as cancer therapies, but also that the dosage
range of a therapeutic IgE antibody which is effective to induce a potent
direct IgE antibody mediated toxicity against the antigen and diseased
cells is also effective for antigen processing, cross presentation and
antigen specific T cell stimulation resulting in a Th1 cell mediated
response (e.g. a CD4, CD8 CTL, IFN gamma associated cellular response).
The IgG therapeutic antibodies of the prior art lack this advantage.
Typically the dosage of an IgG therapeutic antibody required to elicit
the desired effector cell response against the target antigen is orders
of magnitude higher than the dosage needed for antigen cross presentation
and antigen specific T cell stimulation mediated by the IgG therapeutic
antibody. Thus, what is considered the appropriate therapeutic dosage of
a therapeutic IgG antibody is not the optimal dosage for also eliciting a
T cell response by the therapeutic IgG antibody (and if fact is
considered a dosage that is likely to inhibit any T cell response to the
antigen) thereby eliminating the T cell mediated pathway of defense by
the immune system in the treatment of diseases related to the target
antigens. Monoclonal IgE can uniquely utilize direct effector cell
mediated and specific T cell mediated immune pathways at a common dose.
As is used herein the phrase "direct IgE antibody mediated toxicity
against the antigen and diseased cells" means an immune response
involving direct targeting of antigen bearing cells such as ADCC immune
responses, ADCP immune responses or both ADCC and ADCP immune responses
against the antigen/IgE immune complex as evidenced by the stimulation of
eosinophils, mast cells, basophils and other cells to release
pro-inflammatory cytokines, proteases and vasoactive lipid mediators
(e.g. leukotrienes, prostaglandin D2, and platelet activating factor)
when bound to the antigen/IgE immune complex via IgE antibody receptors
FcεRI and FcεRII.

[0053] Therefore, the invention provides methods of increasing the
bioactivity of a therapeutic IgE antibody comprising the step of
administering an IgE antibody to a patient in an amount effective to
induce direct mast cell, basophil, eosinophil effector cell targeting of
the antigen disease site and T-cell mediated cellular immune response to
the antigen in a patient capable of mounting such responses.

[0054] In another example of increasing the bioactivity and biosafety of a
therapeutic IgE antibody of the invention, the rate of administration in
combination with the route of administration can play a significant role
in increasing the biosafety of an antibody of the invention. For example,
in one embodiment, intravenous administration at a concentration of 0.010
to 0.1 mg/ml at a rate of 1 to 4 ml/min for 30 minutes is believed to
increase the biosafety of the therapeutic IgE antibody of the invention
for the reasons discussed below.

[0055] In another example, the monoclonal IgE antibody may administered
intravenously, subcutaneously interarterially or intraperitoneally
depending on the clinical condition of the patient and the location of
the disease process. Direct interarterial administration of the IgE may
also be considered, to assure maximal perfusion of a tumor with the
monoclonal IgE antibody, recognizing that IgE will rapidly clear from the
circulation through the binding of antigen by the antigen-binding region
of the antibody and the binding of effector cells bearing CD23 and
FcεR1. In the case of micro metastatic disease the intravenous
route is generally preferred although in the case of ovarian or other
intra-abdominal cancers, intraperitoneal administration is also a viable
strategy to enhance bioactivity of the IgE antibody in treating
metastatic disease.

[0056] The administration of the therapeutic IgE antibody can be further
enhanced through the pre-administration of a corticosteroid (e.g.
cortisol 50 mg or methyl prodisolone 20 mg) 8 hours to 30 minutes prior
to the IgE antibody infusion. While not interfering with acute immune
stimulatory effects of the antibody administration, the corticosteroid
will reduce the likelihood and severity of any systemic signs and
symptoms associated with the monoclonal IgE infusion.

[0057] Bioactivity is further enhanced by concurrent day administration of
paclitaxel or docetaxel as well as liposomal doxorubicin. For Gemcitabine
Bioactivity is enhanced by administration of the gemcitabine 1 to 4 days
prior to antibody infusion. Chemotherapeutics are to be administered
according to standard dosing protocols for the treatment standards of
specific malignant conditions. Other cytotoxic agents may be administered
according to schedules that must be customized to the clinical case under
consideration.

[0058] Bioactivity and be further enhanced through the scheduled
administration of immune enhancing agents. Such agents include TLR
agonists, for example polyIC or poly IC 12 U which stimulate TLR3
receptors, most notably on antigen processing cells. The TLR agonist may
be given concurrently to 48 hours following antibody administration.
Additional immune stimulatory agents include antibodies that promote
specific T cell immunity, for example anti-CTLA4 or anti-Transforming
Growth Factor-beta dosed concurrent to 48 hours the specific monoclonal
antibody infusion with the intent of preventing the counter regulatory
dampening of specific T cell immunity being stimulated by the monoclonal
IgE.

[0059] The methods according to the invention are useful for treating
patients having a disease associated with an antigen. As used herein, the
term "disease associated with an antigen" means a condition in which
signs or symptoms of illness in a majority of patients are present when
the antigen is present in the patient's body at a certain concentration,
but in which signs or symptoms of illness are absent or reduced when the
antigen is absent from the patient's body or present in the patient's
body at a lower concentration. "Signs or symptoms of illness" are
clinically recognized manifestations or indications of disease. It will
be appreciated that a "patient suffering from a disease associated with
an antigen" of the invention may not yet be symptomatic for the disease.
Accordingly, a patient with circulating CA125 may be is a patient
according to the invention even though that patient may not yet be
symptomatic for ovarian or endometrial adenocarcinoma; and a patient with
elevated PSA may not be symptomatic for prostate cancer. As used herein
the terms "treat," "treating" and "treatment" of a disease associated
with an antigen (e.g. cancer) includes: inhibiting the onset of disease
in a patient; eliminating or reducing tumor burden in a patient;
prolonging survival in a patient; prolonging the remission period in a
patient following initial treatment with chemotherapy and/or surgery;
and/or prolonging any period between remission and relapse in a patient.

[0060] The amount of the composition of the invention which will be
effective in the treatment, inhibition and prevention of the disease
associated with the antigen to which the antibody of the invention is
specific and can be determined by standard clinical techniques. In
addition, in vitro assays may optionally be employed to help identify
optimal dosage ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the judgment
of the practitioner and each patient's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or animal
model test systems.

[0061] For the therapeutic IgE antibodies of the invention, the dosage
administered to a patient is typically 0.001 μg/kg to 1 mg/kg of the
patient's body weight. Preferably, the dosage administered to a patient
is between 0.01 μg/kg and 0.1 mg/kg of the patient's body weight, more
preferably 0.02 μg/kg to 20 μg/kg of the patient's body weight.
Generally, the IgE monoclonal antibodies of the invention have a much
higher affinity for the FCεR (as compared to IgG antibodies, for
example) and longer half-life within the human body than antibodies from
other species. Thus, lower dosages of the antibodies of the invention for
example as much as 100 times lower than typical doses of IgG therapeutic
antibodies and less frequent administration is often possible
particularly when combined with one or more relevant
bioactivity-enhancing agents in accordance with the methods of the
invention.

[0062] As discussed above, the inventors have appreciated that the dose of
monoclonal IgE that will be effective in both mobilizing direct antibody
mediated toxicity against the diseased cells in question and also
mobilize T cell mediated cellular immunity against the disease associated
antigen cell of origin (such as a tumor cell or other antigen source) are
expected to be the same or similar dose and are also expected to be much
lower than the dosages required for direct targeting IgG1 antibodies in
which doses in the range of 10 mg/kg are required. For patients who have
developed treatment preventing immediate hypersensitivity to administered
IgG antibodies, the protocol of and experience of Castells demonstrate
that a starting dose of 0.034 mg/ml can typically be safely infused as a
first dose in a rush desensitization (Castells et al., J. Allergy Clin.
Immunol. 122:574 (2008), Table 1). For a therapeutic IgE antibody of the
invention, a typical therapeutic dose of 1 mg antibody may preferably be
infused as a 0.01 mg/mL solution (100 ml total volume), thus the
therapeutic dose for a therapeutic IgE antibody of the invention is below
the first (and lowest) dose in a rush desensitization protocol of an IgG
antibody. The safe administration of monoclonal IgE thus incorporates a
substantial safety and dosing margin over a standard protocol for
demonstrated limiting hypersensitivity and optionally may include the use
of coadministered bioactivity-enhancing agents to assure the safety
component of the therapeutic index is acceptable.

[0063] In accordance with the methods of the invention, the therapeutic
IgE antibody and the bioactivity-enhancing agent are administered
simultaneously, in either separate or combined formulations, or
sequentially at different times separated by minutes, hours or days, but
in some way act together to provide the desired therapeutic response. As
used herein, "administering" refers to any action that results in
exposing or contacting a composition containing an antibody of the
invention and a bioactivity-enhancing agent with a pre-determined cell,
cells, or tissue, typically mammalian. As used herein, administering may
be conducted in vivo, in vitro, or ex vivo. For example, a composition
may be administered by injection or through an endoscope. Administering
also includes the direct application to cells of a composition according
to the present invention. For example, during the course of surgery,
tumor cells may be exposed. In accordance with an embodiment of the
invention, these exposed cells (or tumors) may be exposed directly to a
composition of the present invention, e.g., by washing or irrigating the
surgical site and/or the cells.

[0064] In one embodiment, the bioactivity-enhancing agent is administered
at least 30 minutes prior to administration of the therapeutic IgE
antibody. In one embodiment the bioactivity-enhancing agent is
administered at least one week prior to, or one week after,
administration of the therapeutic IgE antibody.

[0065] In accordance with a method of the invention compositions
comprising the therapeutic IgE antibody and compositions comprising the
bioactivity-enhancing agent (whether the same or different) may be
administered to the patient by any immunologically suitable route. For
example, the antibody may be introduced into the patient by an
intravenous, subcutaneous, intraperitoneal, intrathecal, intravesical,
intradermal, intramuscular, or intralymphatic routes. The composition may
be in solution, tablet, aerosol, or multi-phase formulation forms.
Liposomes, long-circulating liposomes, immunoliposomes, biodegradable
microspheres, micelles, or the like may also be used as a carrier,
vehicle, or delivery system. Furthermore, using ex vivo procedures well
known in the art, blood or serum from the patient may be removed from the
patient; optionally, it may be desirable to purify the antigen in the
patient's blood; the blood or serum may then be mixed with a composition
that includes a binding agent according to the invention; and the treated
blood or serum is returned to the patient. The invention should not be
limited to any particular method of introducing the binding agent into
the patient.

[0066] Administration may be once, more than once, and over a prolonged
period. As the compositions of this invention may be used for patients in
a serious disease state, i.e., life-threatening or potentially
life-threatening, excesses of the binding agent may be administered if
desirable. Actual methods and protocols for administering pharmaceutical
compositions, including dilution techniques for injections of the present
compositions, are well known or will be apparent to one skilled in the
art. Some of these methods and protocols are described in Remington's
Pharmaceutical Science, Mack Publishing Co. (1982).

[0067] In one preferred embodiment, the bioactivity-enhancing agent is a
corticosteroid such as cortisol (1 mg/kg). Administration of
corticosteroids 8 hours to at least 30 minutes prior to infusion of the
therapeutic IgE antibody will prevent life threatening hypersensitivity
and not interfere with the ability of the patient to generate an antigen
specific T cell response or mediate ADCC or ADCP i.e., generate
protective immunity with anti-antigen IgE-antigen immune complexes.

[0070] In one preferred embodiment the bioactivity-enhancing agent is
paclitaxel and/or docetaxel in combination with a therapeutic IgE
antibody can switch the immune response from Th2 to Th1/Tc1 and enhance
the adaptive immune response. Taxanes signal through toll receptor 4 and
induce TNF-α and IFN-α, mature the dendritic cells and thus
enhance the immune response, especially the Th1 and CTL responses.

[0071] In one preferred embodiment the bioactivity-enhancing agent is a
TLR3 agonist such as poly IC or polylpolyC12U that trigger TNF-α
and IFN-α and IL-6 release in the disease microenvironment, mature
local antigen presenting cells to induce more potent cellular immunity,
and enhance the direct ADCC/ADCP mediated by the IgE coated effector
cells. Timing of TLR administration is concurrent to a window 30 minutes
to several hours following antibody administration. The cytokine
environment induced by TLR is also likely to further inhibit the tendency
for IgE to promote an allergic Th2 driven response as previously observed
by Maurer et al (supra). This may also effect safety as such as switch is
believed to reduce the chance of hypersensitivity.

[0072] In one preferred embodiment the invention provides methods of
enhancing the biosafety of an IgE antibody comprising administering the
antibody intravenously at a concentration of 0.010 to 0.1 mg/ml at a rate
of 1 to 4 ml/min for 30 minutes.

[0073] In another preferred embodiment, the invention provides methods of
enhancing bioactivity by preadministration of corticosteroid; concurrent
administration of a taxane, concurrent to follow up administration of an
immune stimulant such as a TLR agonist, CTLA-4 antagonist, TGF beta
antagonist; and follow up administration by 2 to 7 days of gemcitabine.

[0074] The effectiveness of the methods of the present invention may be
monitored in vitro or in vivo. Humoral responses may be monitored in
vitro by conventional immunoassays, where the anti-tumor activity of the
response may be determined by complement-mediated cytotoxicity and/or
antibody-dependent cellular cytotoxicity (ADCC) assays. The assay
methodologies are well known, and are described in Handbook of
Experimental Immunology, Vol. 2, Blackwell Scientific Publications,
Oxford (1986). Other assays may be directed to determining the level of
the antigen in the patient or tissue. Cell-mediated immunity may be
monitored in vivo by the development of delayed-type hypersensitivity
reactions, or other in vivo or in vitro means known to those skilled in
the art, including but not limited to the skin test reaction protocol,
lymphocyte stimulation assays, measuring the toxicity of a subject's
lymphocytes to tumor cells by using a standard cytotoxicity assay, by a
limiting dilution assay, or by measuring plasma levels of cytokines using
standard ELISA assays.

[0075] Determining the effectiveness of the methods of the invention may
also be accomplished by monitoring cell killing. Those skilled in the art
will recognize that there are a variety of mechanisms that are proof of
cell killing. Cell killing may be demonstrated by showing ADCC, CDC, the
production of natural killer (NK) cells, and/or that cytotoxic T
lymphocytes (CTLs) are produced.

[0076] Human dendritic cells (DC) are cultured from human PBMCs in the
presence of IL-4 and GM-CSF as previously described (Berlyn 2001) and
illustrated in FIG. 1. On day 7, the primary culture is loaded with tumor
associated antigen (TAA) and antibody (Ab) and matured with a standard
maturation cocktail (e.g. TNF-α/IFN-α). The culture is then
combined with autologous lymphocytes and cultured for 7 days.
Subsequently, the lymphocyte culture is stimulated with two additional
rounds of TAA/Ab using fresh dendritic cells cultures as illustrated.
Following the third round of TAA/Ab stimulation, the lymphocytes are
treated with Brefeldin A, which prevents the secretion of synthesized
cytokine. The next day, cells are harvested and stained extracellularly
with anti-CD3-FITC and anti-CD8-PE-Cy5. Cells are then washed, fixed, and
permeabilized and stained intracellularly with anti-IFN-γ-PE and
analyzed by flow cytometry. The CD8.sup.+ IFN-γ producing
population is defined as CD3.sup.+ CD8.sup.+ IFN-γ.sup.+ and the
CD4.sup.+ IFN-γ producing population is defined as CD3.sup.+
CD8.sup.- IFN-γ.sup.+. A representative experiment using an
anti-Her2 IgE, an anti-Her2 IgG1 (Herceptin) and Her2 protein is
illustrated in FIG. 2. Antigen presentation is enhanced by anti-Her2 IgE
and IgG1 Abs. T cells from APA assay were analyzed for IFN-γ
production by intracellular cytokine staining and flow cytometry. T cells
from the APA were stained with anti-CD3-FITC and anti-CD8-PECy5 and then
permeabilized and stained with anti-IFN-γ-PE. Plots are gated on
CD3 (FITC) positive cells and show IFN-γ vs CD8 staining The top
panel is plots of T cells stimulated with PMA and ionomycin (positive
control) or T cells left in media alone (negative control). The bottom
panel includes plots of T cells that were stimulated in the APA with
ECD-Her2 alone (no Ab) or with anti-Her-2 IgE or anti-Her2 IgG1.
Quantification of the effect of the antibody antigen combinations tested
in FIG. 2 are illustrated in FIG. 3, and FIG. 4. The T cell response to
Her2 is normally minimal; however processing of the immune complex with
anti-Her2 IgG1 or anti-Her2 IgE results in a stimulation of antigen
specific T cell immunity with a enhancement of both CD4 and CD8 IFN gamma
phenotypes. Notably IgE to this self antigen induces a potent Tc1
response in contrast to the teachings of the literature based on studies
of allergy. The dose of antigen and antibody is optimal at slight
antibody excess and is similar for IgE mediated primarily through
dendritic cells FcεRII (CD23), as demonstrated via flow
cytometric analysis of an immature and mature in vitro generated DC
population (see FIG. 5) and IgG mediated through Fcγ receptors. In
the illustrated experiment, monoclonal IgE and monoclonal IgG induced a
similar pattern of IFN-γ specific T cell immunity that was not
induced by Ag alone, illustrating the state of tolerance typically
encountered to self- and tumor-Ags.

[0077] An in vitro demonstration of the potential of tumor cells to induce
a local allergic reaction is illustrated using a monoclonal anti-Her2
human IgE with SKBR3 human breast cancer cells or purified Her-2 protein
in conjunction with human fresh leukocyte fraction (targets) incubated
with PBMC or RBL-Sx-38 cells, a rat basophilic leukemia cell line
developed and containing a functional alpha chain to the human
FcεRI (effectors). Tumor cells are incubated with increasing
concentrations of Her-2 specific IgG or IgE and combined with the
effector cells. Evidence of local effector cell activation is measured by
assessing histamine and or beta hexosimimidase release into the culture
supernatant. Confirmatory experiments showing specificity for the Her-2
Ag can be conducted using anti-NP IgE and NP-HSA or anti-PSA IgE and PSA
or conjugated PSA. In addition to immediate hypersensitivity, the target
cells can be pre-labeled with Calcein AM and the ability of the anti-Her2
IgE or IgG to induce tumor killing can be monitored via direct effect of
the PBMC or the RBL-Sx-38 cells in inducing release of the Calcein AM
label.

Example 3

Augmentation of Direct Tumor Targeting

[0078] Having established the baseline activity of a representative Ag
specific IgE monoclonal in terms of both histamine release and basic
tumor cell killing, the effect of corticosteroid and specific adjuvant on
the model can be measured. It will be demonstrated that corticosteroid
does not prevent histamine release; however, it may modestly diminish the
amount of cell killing observed in the assay. The ability of TLR3
stimulation to augment the ADCC effect is addressed by adding low
concentrations (1 to 25 ug/ml) of poly IC (Sigma) to the system at
various time points. The data will reveal that addition of poly IC 30
minutes to 4 hours post co-incubation enhances the amount of killing
observed.

[0079] Passive cutaneous anaphylaxis is performed in transgenic mice
expressing the human FcεRIa. Transgenic mice are shaved and
injected intradermally in different regions of the dorsal side with 50
μA of 6 mg/ml histamine base (HollisterStier, Spokane, Wash.), 1 μg
anti-PSA IgE, CT26-PSA or CT26-Neo microspheroid alone, 2 μg of
crosslinker anti-human kappa antibody alone, 1 μg anti-PSA IgE plus
CT26-PSA or CT26-Neo microspheroid, or 1 μg anti-PSA IgE crosslinked
with 2 μg of an anti-human kappa antibody. After 15 min, 1% Evans Blue
in 250 μl saline is injected i.v. Mice are sacrificed 20 min later,
and local cutaneous anaphylaxis is assessed visually by the blue leakage
in the area surrounding the injection. In the presence of IgE specific
for the tumor cell line; immediate wheal and flare reactions are seen at
the site of the tumor injection, but not in the presence of an antigen
specific IgG antibody or isotype controls. This effect is not prevented
by administration of cortisol to the mouse; however pre-administration of
an H1 blocking agent inhibits the size of the response.

[0080] Macaque monkeys have an FcεR that recognizes human IgE and
the effector cells it is expressed on is similar to those in humans. In
this experiment, Macaque monkeys will be dosed with increasing
concentrations of anti-Her-2 IgE to achieve serum concentrations up to 10
ug/ml. Skin test reactivity to Her-2 protein or glutaraldeyhyde
agglutinated Her-2 protein will be measured by intradermal skin testing.
Administration of complexed Her-2 is expected to induce some signs of
systemic hypersensitivity in the treated monkeys however premedication
with 1 mg/kg cortisone will prevent evidence of systemic anaphylaxis in
the monkeys receiving the infusion of the IgE targeted protein.

Example 6

Tumor Control Studies

[0081] The ability to measure anti-tumor immunity with the human specific
IgE monoclonals in animal models is complicated by the lack of binding of
rodent FcεR to human IgE. In addition to the lack of binding,
mice also see human IgE as a foreign protein and therefore would most
likely mount an immune response against it causing its elimination before
it could be effective in an anti-tumor response. Finally, in order to
perform human tumor studies in mice, the mice also have to be tolerant to
the human tumor antigen. In order to circumvent these obstacles, triple
transgenic mice that express the human FcεRIα chain, human
IgE, and the human tumor antigen of interest (i.e. PSA, Her-2) are
generated. BALB/c mice that express the FcεRIα chain have
been generated and it has been shown that these mice can bind human IgE
via the FcεRI (Dombrowicz, et al., J Immunol 157:1645-51 (1996)).
BALB/c transgenic mice expressing human IgE, human Her-2 and human PSA
are available for cross-breeding to the human FcεRIα
transgenic mice. Pups would need to be tested for the presence of the
transgene to all relevant genes (FcεRIα, human IgE, and PSA
or Her-2). After several round of cross-breeding, triple positive mice
are then selected for immunization experiments in which murine tumor cell
lines (such as CT-26 PSA or SKBR3) containing the human tumor antigen are
injected subcutaneously in the mice. Tumor cell growth is measured in the
absence of antigen specific monoclonal, or in the face of increasing
concentrations of antibody. The ability of TLR stimulation and/or
chemotherapeutic agents, as well as distinct timing of doses of antibody
(Ab) and/or pharmacologic agent, and their effect on the anti-tumor
activity in immunized rodents will also be assessed. Analysis of control
of tumor size and in animal mortality can be used as endpoints.

[0082] The patent and scientific literature referred to herein establishes
the knowledge that is available to those with skill in the art. All
United States patents and published or unpublished United States patent
applications cited herein are incorporated by reference. All published
foreign patents and patent applications cited herein are hereby
incorporated by reference. All other published references, documents,
manuscripts and scientific literature cited herein are hereby
incorporated by reference.

[0083] While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details may be
made therein without departing from the scope of the invention
encompassed by the appended claims. It will also be understood that none
of the embodiments described herein are mutually exclusive and may be
combined in various ways without departing from the scope of the
invention encompassed by the appended claims.